JP2022042503A - Culture vessel and method for producing cultured cell - Google Patents

Abstract

To provide culture vessels that can suppress unexpected movement of cultured cells when a plurality of the culture vessels is stacked and used, and to provide methods for producing cultured cells using the culture vessel.SOLUTION: In a culture vessel 1 having a culture surface 18 and stacked in layers and used, a protrusion 26 that regulates the movement of the stacked culture vessel 1 of the same type in the plane direction is provided on an upper surface 22a of the culture vessel 1, and when each vertex of the rectangle whose area is maximum in the area inside the protrusion 26 on the upper surface 22a is clockwise from point A to point D, the heights HA to HD (mm) of point A to point D measured using a column satisfy 0≤HM/L×100000≤200 (however, HM is the difference in height between the midpoint of the diagonal line AC and the midpoint of the diagonal line BD, and L is the length (mm) of the diagonal line of the rectangle).SELECTED DRAWING: Figure 2

Description

The present invention relates to a culture vessel and a method for producing cultured cells.

Cultured cells are widely used in basic research and drug discovery research to elucidate biological phenomena. In particular, the three-dimensional cell mass (spheroid) in which the cells obtained by the three-dimensional culture are aggregated has a three-dimensional structure similar to that in the living body, so that the test accuracy is improved as compared with the cells obtained by the two-dimensional culture. It is expected to do. As the three-dimensional culture, for example, a culture container (microprocessed container) in which a large number of fine wells (microwells) having a pore diameter of 100 to 1,000 μm were formed on the bottom surface of each well of the microplate, the bottom surface of the dish, or the like was used. A culture method is known (Patent Document 1). When cells are sown in a microfabrication container, the cells associate in each microwell to form a cell mass.

When cell masses pop out of the fine wells due to shaking during culturing or transportation, the cell masses overlap each other, making it difficult to measure or observe with a plate reader, cell imager, or the like. Patent Document 2 proposes stacking and culturing a plurality of culture containers in order to efficiently cultivate plant cells.

Japanese Patent No. 6400575 Japanese Unexamined Patent Publication No. 2011-78886

When a plurality of culture containers are stacked during culturing or transportation, it is difficult to sufficiently suppress shaking. In particular, in order to sufficiently suppress the cell mass from popping out from the fine wells, it is important to minimize the shaking in the state where the culture vessels are stacked.

An object of the present invention is to provide a culture vessel capable of reducing shaking in a state in which a plurality of cells are stacked and suppressing unexpected movement of cultured cells, and a method for producing cultured cells using the culture vessel.

The present invention has the following configurations.
[1] A culture container having a culture surface and used by stacking a plurality of them.
On the upper surface of the culture vessel, a protrusion that regulates the movement of the stacked culture vessels of the same type in the plane direction of the upper surface is provided along the outer edge of the upper surface.
Points A, B, and points clockwise at each apex of a rectangle having a shape similar to the plan view shape of the upper surface of the culture vessel and having the maximum area in a region whose peripheral edge coincides with the inner edge of the upper surface of the protrusion. When C and point D are set
The point A, which is measured in a state where the culture vessel is placed so that the upper surface of the culture vessel faces upward on a support column having a height of 35 mm and a flat upper end surface perpendicular to the height direction. The heights _HA (mm), _BB (mm), HC (mm), and HD (mm) from the lower ends of the columns at points B, _C , and _D are the following formulas 1 and the following formulas. A culture vessel that satisfies 2.
0 ≦ _HM / L × 100,000 ≦ 200 ・ ・ ・ Equation 1
_HM = | ( _HA + HC) / 2- ( _{H B} + _HD ) / 2 |
(However, in the above formula, L is the diagonal length (mm) of the rectangle.)
[2] The shape of the upper surface of the culture vessel in a plan view is a perfect circle, and each vertex of the rectangle having the maximum area in the region of the perfect circle is clockwise as points A, B, C, and D. The culture vessel according to [1].
[3] The culture vessel according to [1], wherein the top surface of the culture vessel has a rectangular shape in a plan view, and the vertices of the rectangular region are clockwise at points A, B, C, and D. ..
[4] The culture vessel according to any one of [1] to [3], wherein a low adhesion coat film formed by applying a cell adhesion inhibitor is formed on the culture surface.
[5] Any of [1] to [4], wherein the material of the culture container is at least one resin selected from polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin and silicone resin. The culture container described in Crab.
[6] The culture vessel according to any one of [1] to [5], wherein a plurality of fine wells are formed on the culture surface.
[7] Using only a plurality of culture vessels according to any one of [1] to [6], stacking a plurality of the culture vessels for either or both of cell culture and transportation after cell culture. A method for producing cultured cells, which is carried out in a state of being in a state of being.

An object of the present invention is to provide a culture vessel capable of reducing shaking in a state where a plurality of cells are stacked and suppressing unexpected movement of cultured cells, and a method for producing cultured cells using the culture vessel. do.

It is a top view which showed the culture container of an embodiment. FIG. 3 is a cross-sectional view taken along the line II of the culture vessel of FIG. It is sectional drawing which showed the state which the plurality of culture containers of FIG. 2 are stacked and used. It is sectional drawing which showed the state of measuring the height of the point A to D of the culture container of FIG. FIG. 3 is an enlarged cross-sectional view showing the culture surface of the culture vessel of FIG. 1. It is a top view which showed the culture vessel of another embodiment. FIG. 6 is a cross-sectional view taken along the line II-II of the culture vessel of FIG. It is sectional drawing which showed the state which the plurality of culture vessels of FIG. 7 are stacked and used. 6 is a cross-sectional view showing how the heights of points A to D of the culture vessel of FIG. 6 are measured. FIG. 6 is an enlarged cross-sectional view showing the culture surface of the culture vessel of FIG. It is sectional drawing which showed the state when 5 culture containers of FIG. 1 were stacked and evaluated. It is sectional drawing which showed the state when 5 culture containers of FIG. 6 were piled up and evaluated.

The meanings and definitions of the terms used herein are as follows.
The numerical range represented by "-" means a numerical range in which the numerical values before and after "-" are the lower limit value and the upper limit value.
The "open end of the fine well" is a boundary line connecting the uppermost portions that orbit the fine well all around. When there is a flat surface around the fine well, the boundary line between the flat surface and the dip of the fine well is the open end of the fine well.
The "opening shape of the fine well" is a plan-view shape of the opening end.
The "diameter of the opening of the fine well" is the diameter of the opening end of the fine well in a plan view, and if the plan view shape of the opening end is not a perfect circle, it is the diameter of the inscribed circle with respect to the plan view shape.
The "opening area of the fine well" is the area of the area occupied by the open end of the fine well in a plan view.
The "depth of the fine well" is the distance between the deepest part of the fine well and the reference surface when the surface that is most in contact with the top that orbits the fine well from above is used as the reference plane. If the perimeter of the fine well is a flat surface, the flat surface coincides with the reference surface.

Hereinafter, an example of an embodiment of the culture vessel of the present invention will be shown and described with reference to the drawings. It should be noted that the dimensions and the like of the figures exemplified in the following description are examples, and the present invention is not necessarily limited to them, and the present invention can be appropriately modified without changing the gist thereof. ..

[First Embodiment]
As shown in FIGS. 1 and 2, the culture container 1 of the present embodiment is a dish including a container main body 10 and a lid portion 20.
The container body 10 is provided on a disk-shaped bottom portion 12, a peripheral wall portion 14 that rises vertically from the outer edge portion of the bottom portion 12 over the entire circumference, and a lower surface 12b of the bottom portion 12 so as to project downward along the outer edge thereof. It is provided with an annular protrusion 16 and an annular protrusion 16 formed therein. The bottom surface of the container body 10, that is, the upper surface 12a of the bottom portion 12 is the culture surface 18. As shown in FIG. 5, a plurality of fine wells 19 are formed on the culture surface 18 of the container body 10 of this example. That is, the culture container 1 is a microfabricated container having a plurality of microwells 19.

The lid portion 20 is formed on the disk-shaped upper surface portion 22, the peripheral wall portion 24 vertically hung from the outer edge portion of the upper surface portion 22 over the entire circumference, and the upper surface portion 22a of the upper surface portion 22 along the outer edge of the upper surface portion 22a. It is provided with an annular protrusion 26 provided so as to project upward over the circumference.

In the culture container 1, the opening above the culture surface 18 of the container body 10 can be freely opened and closed by covering the container body 10 with the lid portion 20. In a state where the lid portion 20 is covered with the container main body 10, the peripheral wall portion 24 of the lid portion 20 is located outside the peripheral wall portion 14 of the container main body 10.

As shown in FIG. 3, a plurality of culture containers 1 can be stacked with the container body 10 covered with the lid portion 20. In a state where a plurality of culture containers 1 of the same type are stacked, the annular protrusion 16 of the container body 10 of the culture container 1 stacked on the upper surface 22a of the lid 20 is fitted inside the protrusion 26 of the lid 20. As a result, the movement of the culture vessel 1 stacked on the upper surface 22a of the lid 20 in the plane direction of the upper surface 22a of the lid 20 is restricted.

The cross-sectional shape perpendicular to the length direction of the protrusion 26 is rectangular in this example, but is semi-circular or the like as long as the movement of the culture vessel 1 stacked on the upper surface 22a of the lid 20 can be regulated. You may.
The protrusions 26 provided on the upper surface 22a of the lid 20 are not limited to the continuous annular protrusions as long as the movement of the culture vessel 1 stacked on the upper surface 22a of the lid 20 in the plane direction can be regulated. , A plurality of protrusions may be formed intermittently.

In the culture vessel 1, the heights of the four points A to D measured by the measuring method described below satisfy a specific condition.
First, as shown in FIG. 1, the area is similar to the plan view shape of the upper surface 22a of the upper surface portion 22 of the culture vessel 1 and the area thereof is within the region E whose peripheral edge coincides with the inner edge 26b of the upper surface 26a of the protrusion 26. Let each vertex of the maximum rectangle be a point A, a point B, a point C, and a point D in the clockwise direction.

In this example, the plan view shape of the upper surface 22a of the upper surface portion 22 is a perfect circle, and the protrusion 26 is formed in an annular shape over the entire circumference along the outer edge of the upper surface portion 22a of the lid portion 20, so that the region E is the region E thereof. The outer periphery is a region of a perfect circle along the inner edge 26b of the upper surface 26a of the protrusion 26. In this region E, the rectangle having the maximum area, that is, the four vertices of the square are clockwise as points A, B, C, and D. All four points A to D are located on the outer periphery of the region E, that is, on the inner edge 26b of the upper surface 26a of the protrusion 26. When the plan view shape of the region E is a perfect circle, the rectangle (square) for determining points A to D is the rectangle (square) having the maximum _HM in the rotation direction with respect to the center of the perfect circle. Is set, and the point where the height from the lower end 112 of the support column 100 is the maximum is set as A.

As shown in FIG. 4, the lid portion 20 of the culture vessel 1 is placed on the support column 100 having a height of 35 mm and a flat upper end surface 110 perpendicular to the height direction so that the upper surface 22a faces upward. Note that FIG. 4 shows a state in which the lid 20 of the culture container 1 is placed on the two columns 100, but if the measurement can be performed in a state where the culture container 1 is stably placed on the columns 100, The number of columns 100 does not have to be two.

In this way, with the lid 20 of the culture vessel 1 placed on the support column 100, the four points A, B, C, and D on the upper surface 26a of the protrusion 26 are respectively from the lower end 112 of the support column 100. The heights of _HA (mm), _BB (mm), _HC (mm), and _HD (mm) are measured.
In the culture vessel 1, the heights HA to HD of the points _A to _D measured in this way satisfy the following formulas 1 and 2.

0 ≦ _HM / L × 100,000 ≦ 200 ・ ・ ・ Equation 1
_HM = | ( _HA + HC) / 2- ( _{H B} + _HD ) / 2 |
However, in the above formula, L is the length (mm) of the diagonal line of the rectangle whose vertices are points A to D.

_HM indicates the difference between the height of the midpoint between the points A and C (the midpoint of the diagonal AC) and the height of the midpoint between the points B and D (the midpoint of the diagonal BD). L is the length of the diagonal AC (= the length of the diagonal BD).

The value represented by _HM / L × 100000 is 0 to 200, preferably 0 to 100, more preferably 0 to 50, still more preferably 0 to 30, particularly preferably 0 to 25, and 0 to 20. Most preferred. When the value represented by _HM / L × 100,000 is within the above range, the strain of the upper surface 22a of the culture container 1 is small, and the rattling when a plurality of culture containers 1 are stacked is reduced. As a result, shaking in a state where a plurality of culture containers 1 are stacked is suppressed. Therefore, when the cell mass is cultured in each fine well on the culture surface, the cell mass is suppressed from popping out from the fine well, which facilitates measurement and observation of the cell mass.

It is preferable that a plurality of fine wells 19 having a uniform size are formed on the culture surface 18 of the container body 10 from the viewpoint that a cell mass having a uniform size can be easily obtained. The example shown in FIGS. 1 and 5 is an embodiment in which there is no flat surface around each fine well 19 on the culture surface 18. The circumference of each fine well 19 on the culture surface 18 may be a flat surface.
The opening shape of the fine well 19 is not particularly limited, and examples thereof include a circular shape, an elliptical shape, a polygonal shape, and an irregular shape.

The average diameter d (FIG. 5) of the opening of the fine well 19 is preferably 100 to 2500 μm, more preferably 200 to 2000 μm, still more preferably 400 to 1000 μm. When the average diameter d of the opening of the fine well 19 is equal to or larger than the lower limit of the above range, a cell mass having a sufficient size is likely to be formed. When the average diameter d of the openings of the fine wells 19 is equal to or less than the upper limit of the above range, the gap between the fine wells becomes small and many fine wells can be efficiently formed on the culture surface, so that the number of cell masses that can be cultured increases. Will increase. The average diameter d of the openings of the fine wells is a value obtained by measuring and averaging the diameters of 10 arbitrary fine wells. The measurement of the diameter of the opening of the fine well can be performed using a planar observation image.

The opening area of the fine well 19 is preferably 0.008 mm ² or more and 4.9 mm ² or less, more preferably 0.03 mm ² or more and 3.1 mm ² or less, and further preferably 0.13 mm ² or more and 0.8 mm ² or less. When the opening area of the fine well 19 is equal to or less than the upper limit of the above range, it is easy to prevent cells from spilling from the fine well 19, and it is easy to form a spheroid having a uniform size.
The opening area of the fine well 19 is measured by a laser microscope (manufactured by KEYENCE CORPORATION) or the like.

The average depth h (FIG. 5) of the fine well 19 is preferably 50 to 1200 μm, more preferably 100 to 1000 μm, and even more preferably 200 to 600 μm. When the average depth h of the fine well 19 is not less than the lower limit of the above range, the cell mass is less likely to pop out from the fine well and is easily held stably. When the average depth h of the fine wells 19 is not more than the upper limit of the above range, bubbles are less likely to be generated in the fine wells when the medium is added, and even if bubbles are generated, bubbles are easily removed. The average depth h of the fine wells is a value obtained by measuring and averaging the depths of the fine wells (distance between the reference plane and the deepest part of the fine wells) for any 10 fine wells. The depth of the fine well can be measured by measuring with a three-dimensional measuring device or by measuring the fine well molded with silicone rubber or the like.

The arrangement pattern of the fine wells 19 is not particularly limited, and may be formed in a regular pattern, irregularly formed, or a mixture of regular portions and irregular portions. As a regular arrangement pattern, for example, a pattern in which fine wells are arranged at each vertex of a square arranged without gaps, a pattern in which fine wells are arranged at each vertex of a regular hexagon arranged without gaps and a fine well in the center, and a staggered pattern are used. It can be exemplified.

The number of fine wells 19 formed on the culture surface 18 is preferably 10 pieces / cm ² or more, more preferably 10 to 10000 pieces / cm ² , still more preferably 15 to 5000 pieces / cm ² , and 20 per unit area. ~ 1500 pieces / cm ² is particularly preferable.

As the material of the culture container 1, resin or glass can be exemplified.
Resins include polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin, silicone resin, polyethylene terephthalate (PET), polyvinyl chloride, high-density polyethylene, polyether sulfan, PET copolymer, and perm. One selected from Knox (Thermofisher Scientific trademark), cycloolefin polymer resin and Cytop (AGC trademark) is preferable, and polystyrene resin is particularly preferable from the viewpoint of high transparency and low drug adsorption. The resin constituting the culture container 1 may be one kind or two or more kinds.
Examples of the glass include quartz glass, borosilicate glass, phosphoric acid glass, and chemically strengthened glass. The glass constituting the culture container 1 may be one kind or two or more kinds.

The peripheral wall portion 14 of the culture vessel 1 may be transparent or opaque. When the peripheral wall portion 14 is opaque, the color tone is more preferably black or white, which makes it difficult for light to pass through. By making the peripheral wall portion 14 opaque, it is possible to reduce the difficulty of observing due to the reflection of light when observing with a microscope. The method for making the peripheral wall portion 14 opaque is not particularly limited, and for example, a method of adding fine particles, a method of adding a coloring agent such as a pigment, or the like can be used. In the case of black, carbon or the like can be used, and in the case of white, titanium oxide or the like can be used.

The average thickness of the bottom portion 12 is preferably 80 μm or more and 2000 μm or less. When the culture vessel 1 is made of glass, the average thickness is more preferably 100 μm or more and 250 μm or less, and further preferably 130 μm or more and 200 μm or less. When the culture vessel 1 is a resin, it is more preferably 300 μm or more and 1800 μm or less, further preferably 500 μm or more and 1500 μm or less, and even more preferably 810 μm or more and 1000 μm or less. When the average thickness of the culture vessel 1 is within the above range, the deformation of the culture vessel 1 due to the formation of the fine wells 19 can be reduced, and the strain during stacking can also be reduced.

The method for producing the culture container 1 is not particularly limited, and can be molded by, for example, an injection molding method.
As a method for forming the fine well 19, for example, laser irradiation can be exemplified. When the culture surface 18 which is the bottom surface of the resin container body 10 is irradiated with laser light, the resin constituting the culture surface 18 is dissolved and vaporized to form fine wells 19 having a very smooth surface. .. Around the opening of the fine well 19, the melted resin may be raised to form a bank portion. In this case, the uppermost portion of the bank portion that goes around the fine well is the open end of the fine well.

The laser light source is not particularly limited, and a CO ₂ laser can be exemplified. The arrangement and size of the fine wells 19 can be adjusted by adjusting the irradiation conditions such as the irradiation position, output, and time of the laser beam.
By fixing the laser output in the range of 1 to 100 W, for example, and fixing the laser irradiation time in the range of 0.1 to 100 μs and performing laser irradiation, the size of each fine well 19 can be made uniform. ..

A low adhesion coat film that suppresses cell adhesion may be formed on the culture surface 18. The formation of a low-adhesion coat film facilitates the removal of cultured cells. The low adhesion coat film can be formed, for example, by applying a cell adhesion inhibitor. Examples of the cell adhesion inhibitor include phospholipid polymers (2-methacryloyloxyethyl phosphorylcholine and the like), polyhydroxyethyl methacrylates, fluorine-containing compounds, and polyethylene glycol. As the cell adhesion inhibitor, one type may be used alone, or two or more types may be used in combination.
If the entire container body 10 or the culture surface 18 of the culture container 1 is molded with a resin having a cell adhesion inhibitory effect such as a silicone resin or a synthetic resin containing the cell adhesion inhibitor, a low adhesion coat film is formed. It is possible to prevent cells from adhering to the culture surface 18 without doing so.

An easy-adhesion coat film may be formed on the culture surface 18 to facilitate the adhesion of cells. Examples of the material for forming the easy-adhesive coating film include collagen and gelatin. As the material for forming the easy-adhesion coating film, one type may be used alone, or two or more types may be used in combination. The entire container body 10 or the culture surface 18 of the culture container 1 may be molded with a resin having a cell adhesion effect, a synthetic resin containing a cell easy adhesion coating agent, or the like.
In addition to the coating agent, physical treatment such as plasma treatment and corona discharge may be performed to facilitate the adhesion of cells.

(Manufacturing method of cultured cells)
Hereinafter, a method for producing cultured cells using the culture vessel 1 will be described.
Only a plurality of culture containers 1 of the same type are used, and one or both of cell culture and transportation after cell culture are performed in a state where a plurality of culture containers 1 are stacked. In the present embodiment, a plurality of culture containers 1 may be stacked only during cell culture, or a plurality of culture containers 1 may be stacked only during transportation, and a plurality of culture containers may be stacked both during cell culture and during transportation. 1 may be piled up.

By providing the protrusion 26 on the upper surface 22a of the lid 20 of the culture container 1, the movement of the culture container 1 stacked on the upper surface 22a of the lid 20 in the surface direction is restricted. Further, since the heights HA to HD of the four points _A to _D in the region along the inner edge 26b of the upper surface 26a of the protrusion 26 satisfy the formula 1 and the formula 2, a plurality of culture containers 1 are stacked. The rattling in the rattling state is reduced and it becomes difficult to shake. From these facts, unexpected movement of cultured cells can be suppressed. Therefore, when a fine well is formed on the culture surface, the cell mass is suppressed from popping out from the fine well, and the measurement and observation of the cell mass become easy.

[Second Embodiment]
As shown in FIGS. 6 and 7, the culture container 2 of the present embodiment is a microplate provided with a container body 30 and a lid portion 40.
The container body 30 rises from the inside of the plate-shaped bottom portion 32 having a rectangular plan view, the peripheral wall portion 34 that rises vertically from the outer edge portion of the bottom portion 32 over the entire circumference, and the peripheral wall portion 34 of the bottom portion 32, and provides each well 35. A plurality of cylindrical tubular portions 36 to be formed, and annular projections 38 provided on the lower surface 32b of the bottom portion 32 so as to project downward over the entire circumference along the outer edge thereof are provided.

In the culture vessel 2 of this example, the bottom surface of each well 35 formed by the bottom portion 32 and the cylinder portion 36 is the culture surface 37. The plurality of wells 35 are arranged vertically and horizontally in a matrix in a plan view. As shown in FIG. 10, a plurality of fine wells 39 are formed on the culture surface 37 of each well 35. That is, the culture vessel 2 is a microfabricated container having a plurality of microwells 39.

The lid portion 40 projects upward along the entire circumference along the upper surface portion 42 having a rectangular plan view shape, the peripheral wall portion 43 vertically hung from the outer edge portion of the upper surface portion 42, and the outer edge of the upper surface portion 42a of the upper surface portion 42. It is provided with a rectangular annular protrusion 44, which is provided so as to be used.

The arrangement pattern of the wells 35 in the culture vessel 2 is not particularly limited, and examples thereof include a matrix shape and a staggered shape. The opening shape of the well 35 in a plan view is not limited to a circle, and may be, for example, a rectangle.
The number of wells 35 included in the culture vessel 2 is not particularly limited, and examples thereof include 1 to 1536.

The average depth of the wells 35 is preferably 6.0 to 12.0 mm, more preferably 5.7 to 10.8 mm. If the average depth of the wells 35 is equal to or greater than the lower limit of the above range, the contents are less likely to spill. When the average depth of the wells 35 is not more than the upper limit of the above range, the solution can be easily dispensed and discharged.

As shown in FIG. 8, a plurality of culture containers 2 can be stacked. In a state where a plurality of culture containers 2 of the same type are stacked, the protrusion 44 provided on the upper surface 42a of the upper surface portion 42 fits inside the annular protrusion 38 of the culture container 2 stacked on the upper surface 42a of the upper surface portion 42. circle. As a result, the movement of the culture vessel 2 stacked on the upper surface 42a of the upper surface portion 42 in the plane direction of the upper surface portion 42a of the upper surface portion 42 is restricted.

The cross-sectional shape perpendicular to the length direction of the protrusion 44 is rectangular in this example, but is semi-circular or the like as long as the movement of the culture vessel 2 stacked on the upper surface 42a of the upper surface portion 42 can be regulated. You may.
The protrusion 44 provided on the upper surface 42a of the upper surface portion 42 is not limited to the continuous annular protrusion as long as the movement of the culture vessel 2 stacked on the upper surface 42a of the upper surface portion 42 in the surface direction can be regulated. , A plurality of protrusions may be formed intermittently.

In the culture vessel 2, the heights of the four points A, B, C, and D measured by the measuring method described below satisfy a specific condition.
First, as shown in FIG. 6, the area is similar to the plan view shape of the upper surface 42a of the upper surface portion 42 of the culture vessel 2 and the area thereof is within the region E whose peripheral edge coincides with the inner edge 44b of the upper surface 44a of the protrusion 44. Let each vertex of the maximum rectangle be a point A, a point B, a point C, and a point D in the clockwise direction.

In this example, the plan view shape of the upper surface 42a of the upper surface portion 42 is rectangular, and the protrusion 44 is formed in a rectangular ring shape over the entire circumference along the outer edge of the upper surface portion 42a of the upper surface portion 42. Therefore, the region E is a rectangular region whose outer circumference is along the inner edge 44b of the upper surface 44a of the protrusion 44. The rectangle having the maximum area in this region E has the same shape as the region E, and the four vertices located at the four corners of the protrusion 44 are clockwise as points A, B, C, and D.

As shown in FIG. 9, the lid portion 40 of the culture vessel 2 and the upper surface portion 42a of the upper surface portion 42 face upward on the support column 100 having a height of 35 mm and a flat upper end surface 110 perpendicular to the height direction. Put it like. As in the first embodiment, the number of columns 100 does not have to be two as long as the heights of points A to D can be measured stably.

In this way, with the culture vessel 2 placed on the column 100, the columns A, B, C, and D on the upper surface 42a of the upper surface 42 of the lid 20 of the culture container 2 are respectively. The heights _HA (mm), _BB (mm), _HC (mm), and _HD (mm) from the lower end 112 of 100 are measured.
In the culture vessel 2, the heights HA to HD of the points _A to _D measured in this way satisfy the formula 1 and the formula 2.

Also in the culture vessel 2, since the value represented by _HM / L × 100000 is 0 to 200, the strain of the upper surface 42a of the upper surface portion 42 of the culture vessel 2 is small, and a plurality of culture vessels 2 are stacked. Time rattling is reduced. As a result, shaking in a state where a plurality of culture containers 2 are stacked is suppressed. Therefore, when the cell mass is cultured in each fine well on the culture surface, the cell mass is suppressed from popping out from the fine well, which facilitates measurement and observation of the cell mass.
The preferred range of values represented by _HM / L × 100,000 is the same as the preferred range of values represented by _HM / L × 100,000 in the first embodiment.

As shown in FIG. 10, it is preferable that a plurality of fine wells 39 having a uniform size are formed on the culture surface 37 of each well 35. There may or may not be a flat surface around each of the fine wells 39 on the culture surface 37. The opening shape of the fine well 39, the average diameter d of the opening (FIG. 10), the opening area, the average depth h (FIG. 10), the arrangement pattern, the number, and the like are the same as those of the fine well 19 of the first embodiment. It can be exemplified, and the preferred embodiment is the same.

As the material of the culture container 2, the same resin and glass as those exemplified as the material of the culture container 1 can be exemplified, and among them, selected from polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin and silicone resin. 1 type is preferable, and polystyrene resin is particularly preferable. Further, the tubular portion 36 may be opaque as in the culture vessel 1.

The average thickness of the bottom portion 32 is preferably 80 μm or more and 2000 μm or less. When the culture vessel 2 is made of glass, the average thickness is more preferably 100 μm or more and 250 μm or less, and further preferably 130 μm or more and 200 μm or less. When the culture vessel 2 is a resin, it is more preferably 300 μm or more and 1800 μm or less, further preferably 500 μm or more and 1500 μm or less, and even more preferably 810 μm or more and 1000 μm or less. When the average thickness of the culture vessel 2 is within the above range, the deformation of the culture vessel 2 due to the formation of the fine well 39 can be reduced, and the strain during stacking can also be reduced.

The method for producing the culture container 2 is not particularly limited, and can be molded by, for example, an injection molding method.
As a method for forming the fine well 39, the same method as the method for forming the fine well 19 can be adopted. A low adhesion coat film that suppresses cell adhesion or an easy adhesion coat film that facilitates cell adhesion may also be formed on the culture surface 37.

(Manufacturing method of cultured cells)
Hereinafter, a method for producing cultured cells using the culture vessel 2 will be described.
Only a plurality of culture vessels 2 of the same type are used, and one or both of cell culture and transportation after cell culture are performed in a state where a plurality of culture vessels 2 are stacked. In the present embodiment, a plurality of culture containers 2 may be stacked only during cell culture, a plurality of culture containers 2 may be stacked only during transportation, and a plurality of culture containers may be stacked both during cell culture and during transportation. 2 may be stacked.

Similar to the case of the culture container 1, the embodiment in which the culture container 2 is used also regulates the movement of the stacked culture containers 2 in the plane direction by the protrusions 44 provided on the upper surface 42a of the upper surface portion 42. Further, since the heights HA to HD of the four points _A to _D in the region along the inner edge 44b of the upper surface 44a of the protrusion 44 satisfy the formula 1 and the formula 2, the stacked culture vessels 2 are loose. Since sticking is reduced and shaking is less likely to occur, unexpected movement of cultured cells can be suppressed. Therefore, when a fine well is formed on the culture surface, the cell mass is suppressed from popping out from the fine well, and the measurement and observation of the cell mass become easy.

The culture vessel of the present invention may be any culture vessel used by stacking a plurality of the culture vessels on the upper surface, and is not limited to a dish such as the culture vessel 1 or a microplate such as the culture vessel 2. The culture vessel of the present invention may be, for example, a flask-shaped culture vessel having an upper surface on which culture vessels of the same type can be stacked. In addition, it is appropriately possible to replace the constituent elements in the above-described embodiment with well-known constituent elements without departing from the spirit of the present invention, and the above-mentioned modifications may be appropriately combined.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following description.
[Stability of stacking]
As shown in FIGS. 11 and 12, five culture vessels (dish or microplate) of the same type are stacked, and the heights (TA to T _D ) of points _A to D of the culture vessels located at the top are measured. Then, the standard deviation was calculated from those measured values, and the stability of stacking was evaluated according to the following criteria.
◎ (excellent): Standard deviation is 0.1 or less.
○ (Good): Standard deviation is more than 0.1 and 0.2 or less.
× (defective): Standard deviation exceeds 0.2.

[Example 1]
In the manner illustrated in FIGS. 1 and 2, four types of culture vessels (Dish-1 to Dish-4 and Dish-6 to Dish-9) having a circular shape with a diameter of 100 mm in a plan view are high. H _A to _HD were measured, and a value represented by _HM / L × 100,000 was calculated. The results are shown in Tables 1 and 2. For Dish-1 to Dish-4, measure the _{heights TA} to TD of the culture vessels located at the top when stacking culture vessels of the same type, calculate the standard deviation, and determine the stability. evaluated. The results are shown in Table 1.

[Example 2]
In the manner illustrated in FIGS. 6 and 7, four types of culture vessels (microplate-1) having a rectangular shape with a plan view shape of 85.3 mm in length × 127.6 mm in width and having 6 (2 × 3) wells. The heights _HA to _HD were measured for the microplate-4 and the microplate-9 to the microplate-11), and the value represented by _HM / L × 100,000 was calculated. The results are shown in Tables 1 and 2. For microplates-1 to microplate-4, measure the _{heights TA} to TD of the culture vessels located at the top when stacking culture vessels of the same type, calculate the standard deviation, and stabilize. Gender was evaluated. The results are shown in Table 1.

[Example 3]
Four types of culture vessels (microplate-5) having 96 (8 × 12) wells in a rectangle having a plan view shape of 85.3 mm in length × 127.6 mm in width in the embodiment as illustrated in FIGS. 6 and 7. -For the microplate-8 and the microplate-12 to the microplate-14), the heights _HA to _HD were measured, and the value represented by _HM / L × 100,000 was calculated. The results are shown in Tables 1 and 2. For microplates-5 to _-7 , measure the heights _TA to TD of the culture vessels located at the top when stacking culture vessels of the same type, calculate the standard deviation, and stabilize. Gender was evaluated. The results are shown in Table 1.

[Example 4]
In the manner illustrated in FIGS. 1 and 2, the heights _HA to H of one type of culture vessel (dish-5, dish-10 and dish-11) having a plan-view shape of a perfect circle with a diameter of 150 mm. _D was measured and a value represented by _HM / L × 100,000 was calculated. The results are shown in Tables 1 and 2. For dish- _{5, the heights TA to T D of} the culture vessels located at the top when stacking culture vessels of the same type were measured, the standard deviation was calculated, and the stability was evaluated. The results are shown in Table 1.

Among the above results, it can be said that the values of _HM / L × 100,000 for Dish-8 and Dish-9 exceed 200, and the distortion of the lid is large, which makes them unsuitable for stacking.

1,2 ... culture container, 10 ... container body, 12 ... bottom, 14 ... peripheral wall, 18 ... culture surface, 19 ... fine well, 20 ... lid, 22 ... top surface, 22a ... top surface, 24 ... peripheral wall, 26 ... protrusion, 26a ... top surface, 26b ... inner edge, 30 ... container body, 32 ... bottom, 34 ... peripheral wall, 35 ... well, 36 ... tube, 37 ... culture surface, 39 ... fine well, 40 ... lid, 42 ... upper surface portion, 42a ... upper surface, 44 ... protrusion, 44a ... upper surface, 44b ... inner edge, 100 ... support, 110 ... upper end surface, 112 ... lower end.

Claims (7)

A culture container that has a culture surface and is used by stacking a plurality of them.
On the upper surface of the culture vessel, a protrusion that regulates the movement of the stacked culture vessels of the same type in the plane direction of the upper surface is provided along the outer edge of the upper surface.
Points A, B, and points clockwise at each apex of a rectangle having a shape similar to the plan view shape of the upper surface of the culture vessel and having the maximum area in a region whose peripheral edge coincides with the inner edge of the upper surface of the protrusion. When C and point D are set
The point A, which is measured in a state where the culture vessel is placed so that the upper surface of the culture vessel faces upward on a support column having a height of 35 mm and a flat upper end surface perpendicular to the height direction. The heights _HA (mm), _BB (mm), HC (mm), and HD (mm) from the lower ends of the columns at points B, _C , and _D are the following formulas 1 and the following formulas. A culture vessel that satisfies 2.
0 ≦ _HM / L × 100,000 ≦ 200 ・ ・ ・ Equation 1
_HM = | ( _HA + HC) / 2- ( _{H B} + _HD ) / 2 |
(However, in the above formula, L is the diagonal length (mm) of the rectangle.) Claimed that the plan view shape of the upper surface of the culture vessel is a perfect circle, and each vertex of the rectangle having the maximum area in the region of the perfect circle is clockwise as points A, B, C, and D. Item 1. The culture vessel according to Item 1. The culture vessel according to claim 1, wherein the top surface of the culture vessel has a rectangular shape in a plan view, and the vertices of the rectangular region are clockwise at points A, B, C, and D. The culture vessel according to any one of claims 1 to 3, wherein a low adhesion coat film formed by applying a cell adhesion inhibitor is formed on the culture surface. The item according to any one of claims 1 to 4, wherein the material of the culture container is at least one resin selected from polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin and silicone resin. Culture container. The culture vessel according to any one of claims 1 to 5, wherein a plurality of fine wells are formed on the culture surface. Using only a plurality of culture vessels according to any one of claims 1 to 6, one or both of cell culture and transportation after cell culture are carried out in a state where a plurality of the culture vessels are stacked. A method for producing cultured cells.

Images